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Objective To investigate the effect of docosahexaenoic acid (DHA) on human colon cancer cell line HT-29 and underlying mechanism. Methods Human colon cancer cell line HT-29 was incubated with DMSO (control), DHA (25, 50, 100 μmol/L) and 100 μmol/L DHA and/or 30 μmol/L 740Y-P. Proliferation was examined by MTT; apoptosis was detected by annexin V-FITC/PI. Western blot was used for detection of protein expression of Bcl-2, Bax apoptosis-related protein and PI3K/Akt/mTOR pathway, and RT-qPCR was used for checking mRNA expression of NLRP3/Caspase-1/IL-1β pathway. Results Compared with the control group, DHA 25, 50,and 100 μmol/L treatment of HT-29 cells resulted in decreased cell survival (P＜0.05), increased apoptosis(P＜0.05), decreased Bcl-2/Bax ratio(P＜0.05) and decreased phosphorylation of PI3K, Akt and mTOR in HT-29 cells(P＜0.05 or P＜0.01). Expressions of NLRP3, Caspase-1 and IL-1β mRNA were decreased (P＜0.05). In addition, cell viability, protein phosphorylation (p-PI3K, p-Akt, p-mTOR)and relative mRNA expression of NLRP3, Caspase 1, and IL-1β were lower in HT-29 cells which were co-incubated with DHA 100 μmol/L and 740Y-P 30 μmol/L than those in the control group (P＜0.05 or P＜0.01) and 740Y-P 30 μmol/L group (P＜0.05), while higher than that of DHA 100 μmol/L group(P＜0.05 or P＜0.01). Conclusions DHA inhibits the proliferation of human colon cancer cell line HT-29, its mechanism is potentially related to the inhibition of PI3K/Akt/mTOR and NLRP3/Caspase-1/IL-1β signaling pathways.